Mitochondria, the centers of cellular energy production, have transferred the majority of their own genetic material to the nuclear genome during evolution. Yet a handful of genes remain in all mitochondrial genomes, despite their susceptibility to damaging metabolic byproducts and mutations. The consequences of mtDNA mutations are significant: they are implicated in a range of severe diseases, and the mutations accumulated during a lifetime are believed to lead to neurodegenerative disorders and the ageing process itself. This raises the question of why the mitochondrial genome still exists, despite the potentially severe consequences on fitness in all eukaryotes, and what are the cellular processes that limit or support mitochondrial gene expression from the nucleus? These questions can be answered by synthetic 'allotopic' expression of these genes from the protected environment of the nucleus. Recent studies have suggested that the lack of success with this strategy is due to the need for adaptations not only in the allotopic protein, but also in several cellular processes. The goal of this project is to systematically study allotopic expression in yeast using a combination of high-throughput and mechanistic biochemical approaches. Yeast is uniquely suited to study this problem because it is one of few organisms where mtDNA can be manipulated, and is amenable to genomic and synthetic biology techniques. Allotopic expression of the 4 yeast genes that have not been experimentally transferred thus far, each of which have been implicated in disease, will be tested in multiple versions by exploiting cost-effective, next-generation oligonucleotide synthesis technology. Applying the power of genetic screens, weakly successful allotopic strains will be used to discover genetic suppressors that improve allotopic expression through genomic screens and in-lab evolution, revealing pathways involved in nuclear gene transfer and mitochondrial biogenesis. These discoveries will be used to produce 'superhost' yeast strains whose backgrounds strongly favour allotopic expression. To discover the roadblocks that prevent allotopic expression and test competing hypotheses for why mtDNA genes have been retained, protein localization, trafficking, susceptibility to degradation, and mitochondrial transport will be tracked. These rewired strains will be characterized at the transcriptomic, bioenergetic, and mechanistic levels. Finally, the allotopically expressed genes will be combined stepwise to generate a strain with a minimal mitochondrial genome. This work will be carried out by leading groups in functional genomics, mitochondrial bioenergetics, and evolution. It will reveal obstacles facing nuclear transfer of mitochondrial genes during evolution, how mitochondrial gene products are expressed and processed, and build a systematic understanding of the key factors in mitochondrial biogenesis. This project will also open new avenues for studying the role of mtDNA in ageing and neurodegenerative disorders.